Achieving Better Sensitivity Less Noise and Fewer Artifacts in NMR Spectra

Both structure determination of large biomolecules by multidimensional NMR and screening techniques require high sensitivity and artifact-free spectra in order to be performed reliably at the lowest possible concentrations. Moreover, automated data analysis software still has not solved the problem of distinguishing artifacts from genuine signals satisfactorily. Hence, the best way to circumvent associated problems is to have as few artifacts or noise as possible. Substantial progress has been...

Adaptation of the Protein Overproducer to the Algal Medium

The described algal hydrolyzate contains amino acids and sugars in a physiological composition, i. e. in a relative composition similar to that required by most host cells. Amino acid biosynthesis, interconversion, and thus the potential for isotope scrambling, are minimized. When all potentially inhibitory low-molecular-weight compounds are removed by extraction prior to hydrolysis, most organisms grow well in media containing their typical salt and trace element composition, with the...

Determination of Protein Dynamics Using 15N Relaxation Measurements

One of the fundamental problems in understanding life at the molecular level is the relationship between structure, dynamics, and function in complex molecular systems like proteins. Proteins are molecular machines that perform most of the functions and control all key events in a living cell. Globular proteins are well packed and adopt ordered three-dimensional structures. Most importantly, they possess a variety of motions such as bond vibrations, side-chain rotations, segmental motions,...

Isotope Labeling and NMR

Recent developments in technologies within structural biology should also play an important part for transmembrane proteins. The potential to incorporate stable isotopes would facilitate structure determination by NMR techniques. Although NMR technologies were long considered to be applicable only to smaller proteins, the development of transverse relaxation-optimized spectroscopy (TROSY) has made it possible to use NMR for larger proteins also 5 , even integral membrane proteins. For example,...

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In two pairs of nuclei A1-A2 and B1-B2, projection angle-dependent cross-correlated relaxation rates due to two dipolar couplings A 2 B1B2 of double and zero quantum coherences between nuclei A1 and B1 can be measured provided the following three requirements are fulfilled (i) The desired double and zero quantum coherence between nuclei A1 and B1 can be excited. (ii) There is a resolvable scalar coupling between A1 and A2 as well as between B1 and B2. For CSA DD cross-correlated relaxation...

Determination of Protein Dynamics Using 15N Relaxation Measurements 283

12.2 Spectroscopic Techniques 284 12.3 Accuracy and Precision of the Method 285 12.3.3 Estimation of Experimental Errors 285 12.5 The Model-Free Approach 289 12.6 Reduced Spectral Densities Mapping 290 12.8 Strategies for the Analysis of Protein Dynamics from 15N Relaxation Data 291 12.10 How Can We Derive the Rotational Diffusion Tensor of a Molecule from Spin-Relaxation Data 293 12.10.1 Theoretical Background 293 12.10.2 Derivation of the Diffusion Tensor when Protein Structure is Known 295...

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Fig. 15.1 Principle of the T1p experiment (A) and of the SLAPSTIC experiment (B). The T1p experiment makes use of the increased transverse relaxation rate of the ligand in the bound state, which leads to slightly reduced signal intensity (A). Signal intensity is drastically reduced or com- pletely quenched by paramagnetic relaxation enhancement from the spin-labeled protein in the case of the SLAPSTIC technique (B). A common spin label is TEMPO C). pletely quenched by paramagnetic relaxation...

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Aggregate Structures of Lipids and their Biophysics Since detergents contain both hydrophobic tails and hydrophilic head groups, they form well-defined aggregates such that the head groups are exposed to the solvent and the hy-drophobic chains are shielded from solvent access. Depending on the relative volumes of head group and aliphatic chains either spherical aggregates, micelles, or bilayers, either vesicles or bicelles, are formed. Micelles spontaneously form when head groups are more bulky...

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Traces through the spectrum c and the evaluation of the desired angle f based on known angles s d are shown as well. Fig. 7.26 _ -resolved constant r C,H-HSQC experiment a to measure the cross-correlated relaxation rates in RNA with a geometry given in b. Traces through the spectrum c and the evaluation of the desired angle f based on known angles s d are shown as well. mined from measurement of the r Cnim in a nondecoupled 13C,1H -HSQC, the circled areas in Fig. 7.26d can be defined for the...

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Fig. 4.1 Schematic diagram of the magnetization arrows or to the a-carbon of the preceding amino transfer pathway in the HNCA experiment. Two acid gray arrows and back to the amide proton pathways exist, that transfer the magnetization for detection. The values of the coupling con- from the amide proton to the amide nitrogen and stants in Hz are indicated next to the arrows. then either to the intra-residual a-carbon black Fig. 4.2 Strips taken from an HNCA experiment of the transcription...

The Cell Free Protein Expression System RTS

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions Scheme 1.1 . All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription translation reaction so that the DNA can be directly used as the template. The first platform RTS 100 HY is designed as a screening...

Stabilizing Proteins by Intein Mediated Backbone Cyclization

Limited protein stability often hampers successful structure elucidation by X-ray crystallography and or NMR spectroscopy. Relaxation properties are usually improved at elevated temperatures, and multidimensional NMR experiments require sample lifetimes to extend over several days to weeks in order to acquire all the necessary data. In addition, the activity of contaminating proteases that are sometimes present in purified samples can be significant at the experimental temperatures. Therefore,...